Identification Of G Protein That Participates In The Regulation Of MTOR Signaling And Screening Of Amino Acids Sensed By Swine T1R1/T1R3 And Study Of The Function

Skeletal muscle is an important organ of animal organism, and is one of the main economical characters. Besides, skeletal muscle is also the main resource of human protein nutrition. Development of skeletal muscle depends on protein deposition, which is the result of protein synthesis and degradation. m TOR is a core regulatory factor of protein synthesis in mammal, and the activity of m TOR is affected by various of signals, including of amino acids, hormones, growth factors, energy status, and so on. GPCR T1R1/T1R3 is an important membrane amino acid sensor, which can sense extracellular amino acids, transmits the signal to the coupled G protein, and finally, regulates the activity of m TOR. Owning to the species-specificity, T1R1/T1R3 in different animals can sense different amino acids. Murine T1R1/T1R3 can sense up to 18 L-amino acids, human T1R1/T1R3 can sense only L-glutmate and L-aspartate, while swine T1R1/T1R3 can sense at most 6 L-amino acids, including L-glutmate, L-aspartate, L-alanine, L-glutamine, L-serine and L-threonine. So amino acids that can regulate m TOR by T1R1/T1R3 will also be defferent in different organisms. According to the issues listed above, we designed two parts of studies, the one is to determine the G protein that couples to T1R1/T1R3 and regulates the activity of m TOR; the second is to screen amino acids that can be sensed by swine T1R1/T1R3, and then verify the function of the amino acids screened out. The main results are:1、Gi2 participates in the T1R1/T1R3-mediated activity regulation of m TOR by amino acid in C2C12 and HEK-293 cells. In C2C12 and HEK-293 cells, activation of Gi2 promotes the phosphorilation of m TOR, p70S6 K and ERK1/2 significantly(P<0.05), which means that Gi2 participates in the regulation of m TOR by affecting the activity of ERK1/2; Besides, Gi2 activation has no effect on the phosphorilation of AKT, which means the regulation of m TOR by Gi2 does not depends on PI3K/AKT signal. Treating with Ca2+ chelator BAPTA-AM inhibits the increase of m TOR, p70S6 K and ERK phosphorilation induced by Gi2 activation significantly(P<0.05), which means the role of Gi2 on m TOR depends on Ca2+ signal. Activation of Gq has no effect on the phosphorilation of m TOR and p70S6 K, which means Gq does not participate in the T1R1/T1R3-mediated avtivity regulation of m TOR by amino acid in C2C12 and HEK-293 cells.2、Swine T1R1/T1R3 can sense Ser and regulate the activity of m TOR. In HEK-293 cells, activation of Gi2 promotes the expression of ERK report plasmid significantly(P<0.05), while treating with different amino acids has no significant effect on the expression of ERK report plasmid; when overexpressing T1R1 and T1R3, incubation of 50mmol/L serine promotes the the expression of ERK report plasmid significantly(P<0.05), which means that swine T1R1/T1R3 can sense serine and regulate the activity of ERK. Besides, incubation of methionine or leucine has no significant effect on on the expression of ERK report plasmid, which means that swine T1R1/T1R3 can not sense methionine and leucine. When overexpressing T1R1 and T1R3, additon of serine will significantly promote the phosphorilation of m TOR, S6K1, and ERK1/2, while addition of 10mmol/L serine has the most significant effect, which means serine can regulate the activation of m TOR by affecting the activity of ERK1/2.In summary, the conclusions of this study are presented as followed:1. T1R1/T1R3 senses amino acid, transmids the signal to the coupled G protein Gi2 and regulates the activity of m TOR. 2. The sense of amino acids by T1R1/T1R3 has species specificity, swine T1R1/T1R3 can sense serine, regulates the activity of ERK and then affects the activation of m TOR.